The long-term objective of this project is to obtain an understanding of the structure and function of the eukaryotic genome. To this end, special attention is being paid to the organization, function and evolution of repeated genes and their gene products. Three repeated genes (or their products) are being studied: the gene for 25S and 17S rRNA, the genes for 5S RNA, and the histone genes. A detailed characterization of the chromatin containing the ribosomal genes (rChr) will be performed. Non-specific endo- and exonucleases and restriction nucleases will be used to characterize the rChr of Tetrahymena which can be labeled specifically using a defined starvation-refeeding regimen. rChr will be isolated and its protein components will be characterized. Tetrahymena 5S genes consist of tandem repeates of 120 bp coding regions alternating with spaces of approximately 160 bp. The organization of these repeating units in macro- and micronuclear chromatin will be probed with non-specific nucleases to determine whether nucleosomes are phased, whether specific genes have specific spacer lengths and whether the DNAse 1 sensitive active genes can be interspersed with DNAse 1 insensitive regions less than 1 nucleosome in lengths. The role of histones and their modifications in maintaining the structural and functional differences between Tetrahymena macronuclei (genetically active) and micronuclei (genetically inert) will be studied. The sequence of Tetrahymena histones and of their sites of secondary modification will be examined to determine the relationships between functional and evolutionary divergence.